The goal of this project is to uncover the physical basis for the ion-mediated interactions of retroviral RNA kissing-loop complexes and to explain the unusual sequence requirements for maximum mechanical stability;specifically that of the Dimerization Initiation Site (DIS) of HIV and Moloney Leukemia Virus (MMLV). This high stability is known to be crucial for retroviral genome dimerization, as mutations to the DIS loop always result in greatly reduced virus replication and infectivity rates in vivo. Therefore, interfering with kissing-loop mediated genome dimerization may prove to be a successful route to designing new anti-retroviral therapeutics. However, current attempts to target this interface have actually resulted in increased kissing-loop stability with no detectible inhibition of viral replication. It would therefore be useful to determine the physical basis of the enhanced kissing loop stability in order to inform future attempts at designing targeted inhibitors. Mutational analysis has shown that several bases flanking the loop residues are crucial for high complex stability, but both structural and chemical mapping experiments confirm that these bases are not base paired, do not participate in intra or inter-molecular hydrogen bonds, and actually appear to be flipped out into solution. Lastly, the observed separation distances at the transition state are too large to be explained by partial base-pairing or the presence of interstitial water molecules. We hypothesize that the flanking residues effect neigboring base pair dissociation kinetics through modulation of the local ionic environment. We also predict that the release of partially dehydrated ions at the transition state constitutes the rate-limiting step for kissing-loop dissociation. Using explicit ion, implicit solvent Monte Carlo simulations, the kinetic pathways of kissing loops dissociation will be determined. A Markov state model of the dominant dissociation pathway will be created, and then examined in detail using large numbers of short, all-atom molecular dynamics simulations of transitions along dissociation intermediates. These simulations will utilize an applied external force to enhance dissociation, analagous to single-molecule pulling experiments. The accuracy of the simulations will be ascertained by comparison of the predicted force-extension curves, separation at the transition state, critical force, and dissociation rates with the actual experimental measurements. In this way, the extent to which the unpaired flanking residues indirectly contribute to the overall mechanical stability of the complex through ion-mediated modulation of base pairing kinetics at the loop-loop interface will be ascertained. Understanding ion-mediated driving forces for complex formation should allow better prediction of stabilizing and destabilizing mutations, as well as identify specific ion-mediated interactions which may be exploitable in the design of small molecule inhibitors. PUBLIC HEALTH RELEVANCE: This aims of this research is to provide physical insight into the unexplained strength of a kissing- loop motif that is absolutely required for replication of the HIV retrovirus. Identification of the specific interactions that give rise to enhanced kissing-loop stability should aid in the design of new anti-retroviral drugs.